Electrophoresis is a well known separation technique which separates charged units on the basis of differential mobility in an electric field (which depends on the size, shape and charge of the units), and is widely used for separation of proteins and other materials. A variant of electrophoresis known as isoelectric focusing (IEF) exploits the fact that the net charge on a protein molecule varies with the pH of the surrounding solution. At a pH that is characteristic for each protein there exists an isoelectric point (IEP) at which the protein has no net charge and therefore will not migrate in an electric field. In IEF, electrophoresis is carried out in a pH gradient established by use of buffer mixtures, commonly using carrier ampholytes, and each protein migrates to the position in the gradient that corresponds to its isoelectric point and then remains there.
Two dimensional (2D) electrophoresis techniques are also known, involving a first electrophoretic separation in a first dimension, followed by a second electrophoretic separation in a second, transverse dimension. The 2D method most commonly used is that based on the work of O'Farrell (reference 1) in which proteins are subjected to IEF in a polyacrylamide gel in the first dimension, resulting in separation on the basis of isolectric point, and are then subjected to polyacrylamide gel electrophoresis in the second dimension in the presence of sodium dodecyl sulphate (SDS), resulting in further separation on the basis of size.
Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) is at present the most highly resolving method for the analysis of protein mixtures. Using this technique it is possible to separate several thousand individual polypeptide chains from a single sample in a single electrophoretic analysis. Methods which enable this are well established, used widely and all of the necessary equipment and reagents can be obtained readily from commercial sources.
Proteins are detected usually by staining either by a dye, which is most commonly Coomassie Brilliant Blue R-250 (C.I. 42660), or by deposition of metallic silver. Alternatively proteins which have been radiolabelled before analysis either in vivo or in vitro, for instance by reductive methylation (reference 2), can be detected by autoradiography or fluorography. The radiolabelling method is the only one which is at present available in which proteins can be labelled before analysis by 2D-PAGE in which IEF is used as the first dimension.
Fluorophores have also been used to label proteins both before and after PAGE. Several papers have described prelabelling methods for 1D-PAGE (reference 3). The advantages of pre- over post-electrophoretic labelling are the possibility of viewing the separation during the electrophoresis, the ease of detecting the results at the end without further processing of the gel, the ease of viewing gels of various sizes and thickness, the avoidance of problems associated with handling delicate gels, the avoidance of losing small molecules from the gel during staining, the high sensitivity which can be achieved, and the avoidance of use of radioactive materials.
Recently the fluorophore labelling of proteins present in IEF gels has been described which enabled the subsequent generation of a fluorescent 2D-electrophoretogram. The method allows normal IEF to be combined with the advantages of pre-electrophoretic fluorescent labelling (reference 4).
Another recent paper (reference 5) describes in vivo monitoring of protein sulphydryl (SH) groups of hamster spermatazoa by labelling with the fluorescent material monobromobimane and 2-D electrophoretic analysis. The first dimension uses non-equilibrium pH gradient electrophoresis (NEPHGE), followed by SDS-PAGE in the second dimension. In NEPHGE a pH gradient is generated but proteins are loaded at the acidic end of the gel, are positively charged and do not reach their IEPs. The method is used for analysing proteins with basic IEPs which cannot be analysed by IEF because of the difficulty in obtaining stable pH gradients at alkaline pHs.
Hitherto, no method has been demonstrated which enables useful separations to be obtained after the pre-IEF labelling of proteins with either fluorophores or chromophores. So far no protein derivatization with a fluorophore or chromophore has been demonstrated in which the charge properties (ie pK) of the derivative exactly mimics that of the underivatized protein. Thus, derivatization with such reagents causes changes in the IEPs of the proteins, which leads to the IEF pattern being altered. The reason for this is two fold. It can be difficult to ensure 100% modification of the protein. Partial modification will lead to the focusing at different IEPs of each species of each individual polypeptide carrying a different percentage of modification. Indeed, this property of partially modified proteins has been used to generate a "charge-train", by carbamylation of lysine residues by cyanate. Such charge modifications can be used as a standard for IEF (eg Carbamylyte (Pharmacia-LKB)). Moreover, if 100% modification of one type of reactive residue (eg lysine) is obtained and the charge on it altered then the protein will focus with a new IEP and thus the pattern of separation will be either quite different from that obtained with unmodified protein or it may be impossible to obtain a useful separation pattern (eg if the protein becomes very acidic).
The present invention is based on a novel approach to labelling of proteins for separation by IEF, which enables separating by IEF of fluorescently pre-labelled proteins.